Brassica rapa Nitrate Transporter 2 (BrNRT2) Family Genes, Identification, and Their Potential Functions in Abiotic Stress Tolerance

Nitrate transporter 2 (NRT2) proteins play vital roles in both nitrate (NO3−) uptake and translocation as well as abiotic stress responses in plants. However, little is known about the NRT2 gene family in Brassica rapa. In this study, 14 NRT2s were identified in the B. rapa genome. The BrNRT2 family members contain the PLN00028 and MATE_like superfamily domains. Cis-element analysis indicated that regulatory elements related to stress responses are abundant in the promoter sequences of BrNRT2 genes. BrNRT2.3 expression was increased after drought stress, and BrNRT2.1 and BrNRT2.8 expression were significantly upregulated after salt stress. Furthermore, protein interaction predictions suggested that homologs of BrNRT2.3, BrNRT2.1, and BrNRT2.8 in Arabidopsis thaliana may interact with the known stress-regulating proteins AtNRT1.1, AtNRT1.5, and AtNRT1.8. In conclusion, we suggest that BrNRT2.1, BrNRT2.3, and BrNRT2.8 have the greatest potential for inducing abiotic stress tolerance. Our findings will aid future studies of the biological functions of BrNRT2 family genes.


Introduction
Nitrogen is an important element for the growth and development of plants, as it plays a significant role in shaping the composition of proteins and nucleic acids.Furthermore, nitrogen is a key factor affecting both the yield and quality of crops.Within the soil environment, the forms of nitrogen that plants utilize include ammonium nitrogen (NH 4 + ) and nitrate nitrogen (NO 3 − ).Nitrate is the primary nitrogen source for most dryland crops, including wheat, soybean, and Chinese cabbage.Its uptake and transport are facilitated by nitrate transport proteins.Nitrate transporter 2 (NRT2) is part of the NITRATE/NITRITE PORTER (NNP) family, which in turn belongs to the MAJOR FACILITATOR SUPERFAMILY (MFS).The structure of the NRT2 protein generally includes 500-600 amino acids (aa) and contains 12 transmembrane helical segments [1].This protein was first discovered and characterized in Aspergillus nidulans [2].Molecular cloning of the NRT2 gene in higher plants was first reported in barley [3].NRT2 family members are categorized as high-affinity transport system (HATS), and most of the family members need to bind to the molecular chaperone nitrate-assimilation-related 2 (NAR2 or NRT3) to activate their high-affinity transport activity, which is important when there is a low concentration of exogenous nitrate; NRT2 family members, thus, play a key role in plant growth and development.
Several studies have reported the functions and evolutionary history of NRT2 genes in different plant species.NRT2s are responsible for nitrate absorption and transport in plants.In A. thaliana, seven members of the NRT2 family have been identified [4].AtNRT2.1 performs a key dual role in controlling root development with external NO 3 − availability [5], and AtNRT2.2 and AtNRT2.1 synergistically regulate NO 3 − uptake and the dynamic response of plants to changes in the environmental nitrogen content [6].
Genes 2023, 14 AtNRT2.4plays a dual role in the shoots and roots of nitrogen-starved plants [7].AtNRT2.5 is necessary to support the growth of nitrogen-starved mature plants by working with AtNRT2.1,AtNRT2.2, and AtNRT2.4,which ensures the efficient nitrate uptake, and by facilitating nitrate loading into the phloem during nitrate remobilization [8].AtNRT2.7 is mainly responsible for the loading of nitrate in seed vacuoles and plays a role in NO 3 − storage [9].In bread wheat (Triticum aestivum L.), TaNRT2.1 is involved in nitrate uptake at the post-flowering stage [10].In chrysanthemum (Chrysanthemum morifolium), CmNRT2.1 is a nitrate-inducible gene, and its activity can affect nitrate uptake [11].In pineapple (Ananas comosus), AcNRT2.2 is highly expressed in the roots, suggesting that AcNRT2.2might have a significant impact on alleviating nitrate deficiency [12].NRT2s also play a role in mediating resistance to biotic and abiotic stress.In A. thaliana, AtNRT2.1 has been shown to regulate root hydraulic conductivity and plasma membrane aquaporin activity [13], suggesting that it may enhance plant drought resistance and other processes related to root hydraulic conductivity.Additionally, AtNRT2.1 works as a significant contributor to Cd uptake by modulating nitrate uptake in high-affinity nitrate systems [14], and NRT2.1 can also affect plant disease resistance by downregulating biotic stress defense mechanisms and promoting abiotic stress responses [15].AtNRT2.6 expression is induced after inoculation of A. thaliana with the phytopathogenic bacterium Erwinia amylovora.The reduced expression of NRT2.6 can reduce pathogen tolerance, and its activity is likely linked to ROS production under biotic and abiotic stress [16].In B. napus (Brassica napus L.), a total of seventeen members were identified.BnNRT2.1a,BnNRT2.5s, and BnNRT2.7swere found to be involved in the response to waterlogging stress.BnNRT2.7splays an important role under P-and K-deficient conditions [17].In tomato (Solanum lycopersicum), four NRT2 members have been identified to positively affect the response of plants to drought and salt stress [18].
In addition, NRT2s also regulate the transport of auxin to the root system of plants to participate in nitrate-dependent root elongation [19].They also play a role in regulating the control of cytokinins [20] and are involved in the biosynthesis and signal transduction of ethylene [21].Their significance is further supported by their contribution to the root morphogenesis of A. thaliana [5].They enhance the pH-buffering capacity of plants [22] and promote the uptake of manganese [23] and phosphorus in rice [24].
Chinese cabbage (B.rapa) is a biennial herb of the genus Brassica in the Cruciferae family that is widely grown in Asia.It thrives in mild climates and soils with abundant moisture and nitrogen fertilizer.However, B. rapa exhibits low nitrogen utilization and frequently faces challenges such as drought and salt stress, which significantly hinders its yield and quality [25,26].An increasing number of studies indicate that processes involved in nitrogen assimilation, metabolism, and transport are intimately tied to drought and salt stress in plants.For example, the adverse effects of drought stress on Malus prunifolia can be mitigated when the nitrogen supply is robust [27].In the case of oil palm, reactive nitrogen metabolic activities and nitrate assimilation processes contribute to the response of plants to drought stress [28].Additional studies on sorghum and tomato revealed that the introduction of exogenous nitrogen could notably alleviate the intake of Na + and bolster the K + content in these plants [29,30].There is, thus, a need to clarify the roles and molecular mechanisms of related genes.This knowledge is important for enhancing the yield and quality of B. rapa and strengthening its resilience to drought and salt stress.In this study, we obtained basic information on BrNRT2 genes and their expression profiles after drought stress and salt stress treatments.The insights gained from this study will contribute to future studies aimed at clarifying the functions of NRT2 genes.

Phylogenetic Tree Construction and Collinearity Analysis
The aa sequences of BrNRT2s, AtNRT2s, OsNRT2s, PtNRT2s, and HvNRT2s were aligned using the MUSCLE algorithm in MEGA X (v11.0.13), and a phylogenetic tree was constructed using the maximum likelihood method (model: LG + G) in MEGA X with 1000 bootstrap replicates to ensure the accuracy and reliability of our results [34].The phylogenetic tree was visualized using the online software iTOL (v6.8) [35] (https: //itol.embl.de/,accessed on 1 May 2023).To explore the genetic homology across different plant species, we downloaded the genomic and annotation information files of Arabidopsis, soybean, and potato from the EnsemblPlants database (release 56) (http://plants.ensembl.org/, accessed on 1 May 2023).The MCScanX (Multiple Collinearity Scan toolkit) plug-in in TBtools (v1.120) was used to analyze interspecies collinearity.

Gene Structure and Conserved Domain Analysis
The structure of BrNRT2s was visualized using the Visualize Gene Structure program of TBtools (v1.120) [31].

Cis-Regulatory Elements, and GO Enrichment Analysis
TBtools (v1.120) was used to extract the 2.0 kb promoter sequence upstream of the start codon of BrNRT2s.The characteristics of the cis-regulatory elements were predicted using the PlantCARE (v1) (http:/bioinformatics.psb.ugent.be/webtools/plantcare/html/,accessed on 3 May 2023) [38] with the default parameters.
In addition, to analyze NRT2s based on their functional similarity, we performed GO enrichment analysis using the online DAVID database (v2023q1) [39] (https://david.ncifcrf.gov/, accessed on 3 May 2023), and the GO annotation data were processed and graphically demonstrated using bioinformatics (http://www.bioinformatics.com.cn/,accessed on 3 May 2023).
The first-generation hybrid cultivar of B. rapa with stable self-incompatibility was used for stress treatments.Plump seeds were seeded in MS Modified Medium (with vitamins, Sucrose, Agar) (PM10121-307, Coolaber, Beijing, China) and cultivated in a plant incubator (16 h light/8 h dark photoperiod at 25 • C, light intensity 2000 lx).When the seedlings were 4 weeks old, seedlings with similar growth conditions were subjected to stress treatments.These seedlings were immersed in a 150 mmol•L −1 NaCl solution prepared with halfstrength Hoagland nutrient solution (pH = 5.8) to simulate salt stress; they were also immersed in 15% PEG6000 to simulate drought conditions, whereas normal hydroponic seedlings were used as the control (the hydroponic pots had a volume of 10 L).All stress treatments were administered for 0, 4, 6, and 12 h for a total of 4 treatments.Each treatment consisted of three biological replicates.The leaves and roots of B. rapa under drought and salt stress were sampled and used for RNA extraction and qRT-PCR analysis.
The STRING website (v11.5)(https://cn.string-db.org/,accessed on 12 May 2023) was used to predict protein-protein interaction (PPI) relationships [43].In this study, we selected "A.thaliana" as the focal organism and used the "single protein by sequence" method.Specifically, we searched for A. thaliana NRT2 proteins in the STRING database that were similar to BrNRT2.1,BrNRT2.3, and BrNRT2.8aa sequences using the BLASTP method.From this analysis, we selected the most similar proteins to establish a comprehensive protein interaction network diagram.

Identification, Physicochemical Characterization, Chromosomal Localization, and Subcellular Localization of BrNRT2s
A total of 14 BrNRT2 genes were screened from the B. rapa genome, and they were unequally distributed on 6 of the 10 chromosomes of B. rapa (Figure 1).There were two BrNRT2 genes on chromosomes 1, 2, 8, 9, and 10.Four genes were mapped to chromosome 6.They were named BrNRT2.1~BrNRT2.14(Table 1) according to their sequences on the chromosomes.To investigate the physicochemical properties of these BrNRT2 genes, we used ExPASy online software and found that the isoelectric point (pI) of the proteins ranged from 6.89 (BrNRT2.7) to 9.15 (BrNRT2.4).The number of basic proteins (12) was significantly greater than the number of acidic proteins (2).The molecular weights of the proteins ranged from 52,132.17Da (BrNRT2.3) to 62,619.10Da (BrNRT2.9).The lengths of the BrNRT2 proteins ranged from 484 aa (BrNRT2.3) to 575 aa (BrNRT2.9).The subcellular localization analysis of BrNRT2s showed that all BrNRT2 proteins were distributed in the plasma membrane (plas), suggesting that these proteins might perform their functions on the plasma membrane and that they potentially mediate nitrate transport.plasma membrane (plas), suggesting that these proteins might perform their functions on the plasma membrane and that they potentially mediate nitrate transport.

Phylogenetic Tree Construction and Collinearity Analysis
To better understand the evolutionary features among NRT2 family members, a phylogenetic tree based on sequence similarity was created for the NRT2 family genes from diverse plant species using the maximum likelihood method (Figure 2A).BrNRT2 genes
To gain further insights into the origin and evolution of BrNRT2 genes, we examined the syntenic relationships between B. rapa and three other plants (A.thaliana, G. max, and S. tuberosum).There were nine collinear pairs of BrNRT2 genes and AtNRT2 genes.There were six collinear pairs of BrNRT2 genes and GmNRT2 genes and six collinear pairs of BrNRT2 genes and StNRT2 genes.These observations suggest that B. rapa and A. thaliana are evolutionarily closely related and functionally similar; these findings aid exploration of the functions of BrNRT2s (Figure 2B).
the syntenic relationships between B. rapa and three other plants (A.thaliana, G. max, and S. tuberosum).There were nine collinear pairs of BrNRT2 genes and AtNRT2 genes.There were six collinear pairs of BrNRT2 genes and GmNRT2 genes and six collinear pairs of BrNRT2 genes and StNRT2 genes.These observations suggest that B. rapa and A. thaliana are evolutionarily closely related and functionally similar; these findings aid exploration of the functions of BrNRT2s (Figure 2B).
To predict the conserved domains of NRT2 proteins in B. rapa (Figure 3B), we utilized NCBI-CDD and identified two functional domains present in this gene family: PLN00028 and MATE_like superfamily.BrNRT2.9 has a unique combination of PLN00028 and MATE_Like superfamily domains, suggesting that this gene potentially has special functions.The other 13 family members only have PLN00028 domain.

Cis-Elements Analysis
To further investigate the mechanism of the BrNRT2 genes in response to abiotic stress, the 2 kb promoter sequences upstream of the start codon of BrNRT2s were analyzed (Figure 4).Each member of the BrNRT2 family contains abundant light-responsive elements, which emphasizes the key role of light signal regulation in the growth and development of B. rapa.Additionally, all members contain phytohormone response elements; approximately 90% of the BrNRT2 genes contain ABA elements, and approximately 85% of BrNRT2 genes contain methyl jasmonate (MeJA)-responsive elements.With the exception of BrNRT2.7 and BrNRT2.12,all members contained cis-regulatory elements related To predict the conserved domains of NRT2 proteins in B. rapa (Figure 3B), we utilized NCBI-CDD and identified two functional domains present in this gene family: PLN00028 and MATE_like superfamily.BrNRT2.9 has a unique combination of PLN00028 and MATE_Like superfamily domains, suggesting that this gene potentially has special functions.The other 13 family members only have PLN00028 domain.

Cis-Elements Analysis
To further investigate the mechanism of the BrNRT2 genes in response to abiotic stress, the 2 kb promoter sequences upstream of the start codon of BrNRT2s were analyzed (Figure 4).Each member of the BrNRT2 family contains abundant light-responsive elements, which emphasizes the key role of light signal regulation in the growth and development of B. rapa.Additionally, all members contain phytohormone response elements; approximately 90% of the BrNRT2 genes contain ABA elements, and approximately 85% of BrNRT2 genes contain methyl jasmonate (MeJA)-responsive elements.With the exception of BrNRT2.7 and BrNRT2.12,all members contained cis-regulatory elements related to abiotic stress, including low-temperature-responsive elements, drought-inducibility elements, defenseand stress-responsive elements, and anaerobic induction elements.These results indicate that members of the BrNRT2 gene family play a fundamental role in enhancing abiotic stress tolerance in plants.
to abiotic stress, including low-temperature-responsive elements, drought-inducibility elements, defense-and stress-responsive elements, and anaerobic induction elements.These results indicate that members of the BrNRT2 gene family play a fundamental role in enhancing abiotic stress tolerance in plants.

GO Enrichment Analysis
GO analysis on the BrNRT2 genes contributed to determining their functions.GO analysis showed that BrNRT2 genes were enriched in biological process (BP), cellular component (CC), and molecular function (MF) (Figure 5; Table S2).Based on a previous study, nitrate transporters play an essential role in drought and salt tolerance [18].The three enriched terms in the BP category were nitrate transport (GO:0015706), cellular response to nitrate (GO:0071249), and nitrate assimilation (GO:0042128).This prompted us to further characterize the expression changes of BrNRT2 family members under drought and salt stress conditions.The three enriched terms in the CC category were plant-type vacuole membrane (GO:0009705), plasma membrane (GO:0005886), and integral component of membrane (GO:0016021).The GO-MF enrichment results revealed one enriched term: nitrate transmembrane transporter activity (GO:0015112).Two BrNRT2 genes were enriched in the nitrate assimilation term; nine genes were enriched in nitrate transport and cellular response to nitrate, albeit with a small p-value but high confidence; and nine genes were enriched in plant-type vacuole membrane, plasma membrane, integral component of membrane, and nitrate transmembrane transporter activity.

GO Enrichment Analysis
GO analysis on the BrNRT2 genes contributed to determining their functions.GO analysis showed that BrNRT2 genes were enriched in biological process (BP), cellular component (CC), and molecular function (MF) (Figure 5; Table S2).Based on a previous study, nitrate transporters play an essential role in drought and salt tolerance [18].The three enriched terms in the BP category were nitrate transport (GO:0015706), cellular response to nitrate (GO:0071249), and nitrate assimilation (GO:0042128).This prompted us to further characterize the expression changes of BrNRT2 family members under drought and salt stress conditions.The three enriched terms in the CC category were plant-type vacuole membrane (GO:0009705), plasma membrane (GO:0005886), and integral component of membrane (GO:0016021).The GO-MF enrichment results revealed one enriched term: nitrate transmembrane transporter activity (GO:0015112).Two BrNRT2 genes were enriched in the nitrate assimilation term; nine genes were enriched in nitrate transport and cellular response to nitrate, albeit with a small p-value but high confidence; and nine genes were enriched in plant-type vacuole membrane, plasma membrane, integral component of membrane, and nitrate transmembrane transporter activity.

Tissue-Specific Expression
To investigate the expression of the BrNRT2 genes in various tissues of B. rapa, we obtained tissue-specific transcriptome data from the Brassicaceae Database.The transcriptome data of BrNRT2s extracted from five different tissues (root, stem, flower, leaf, and silique) (Figure 6; Table S3) revealed that the expression levels of BrNRT2.1,BrNRT2.5, BrNRT2.6,BrNRT2.8,BrNRT2.10,BrNRT2.11,BrNRT2.12, and BrNRT2.13 were significantly higher in the roots than in the other tissues, indicating that these genes may play an important role in nitrate uptake from the soil and in the response to root-associated stresses.BrNRT2.4 and BrNRT2.14 were only highly expressed in the stems.These genes, which were specifically expressed in a single tissue, may play a role in loading and discharging nitrate.BrNRT2.2 and BrNRT2.7 were highly expressed in stem and flower tissues.BrNRT2.3 was highly expressed in stem and leaf tissues, and BrNRT2.9 was highly expressed in root, stem, and leaf tissues, suggesting that these genes may play a role in the long-distance transport of nitrate from the roots to the aboveground parts.The expression of all genes in the callus was not significant.These tissue-specific expression differences among the BrNRT2 genes suggest that they play distinct roles in different stages of plant development.

Tissue-Specific Expression
To investigate the expression of the BrNRT2 genes in various tissues of B. rapa, we obtained tissue-specific transcriptome data from the Brassicaceae Database.The transcriptome data of BrNRT2s extracted from five different tissues (root, stem, flower, leaf, and silique) (Figure 6; Table S3) revealed that the expression levels of BrNRT2.1,BrNRT2.5, BrNRT2.6,BrNRT2.8,BrNRT2.10,BrNRT2.11,BrNRT2.12, and BrNRT2.13 were significantly higher in the roots than in the other tissues, indicating that these genes may play an important role in nitrate uptake from the soil and in the response to root-associated stresses.BrNRT2.4 and BrNRT2.14 were only highly expressed in the stems.These genes, which were specifically expressed in a single tissue, may play a role in loading and discharging nitrate.BrNRT2.2 and BrNRT2.7 were highly expressed in stem and flower tissues.BrNRT2.3 was highly expressed in stem and leaf tissues, and BrNRT2.9 was highly expressed in root, stem, and leaf tissues, suggesting that these genes may play a role in the long-distance transport of nitrate from the roots to the aboveground parts.The expression of all genes in the callus was not significant.These tissue-specific expression differences among the BrNRT2 genes suggest that they play distinct roles in different stages of plant development.

Effect of Drought Stress on the Expression of BrNRT2s
Drought stress can negatively affect B. rapa production.To gain a preliminary und standing of the function of BrNRT2 genes in response to drought stress, we obtained transcriptome sequencing data of drought-sensitive (DS) B. rapa from the Brassicaceae D tabase (Figure 7A) and used RNA-seq to detect the expression levels of BrNRT2 genes af drought stress (Figure 7B).Three genes (BrNRT2.1,BrNRT2.3, and BrNRT2.10)with h expression levels in B. rapa were selected for further qRT-PCR detection (Figure 7C).T

Effect of Drought Stress on the Expression of BrNRT2s
Drought stress can negatively affect B. rapa production.To gain a preliminary understanding of the function of BrNRT2 genes in response to drought stress, we obtained the transcriptome sequencing data of drought-sensitive (DS) B. rapa from the Brassicaceae Database (Figure 7A) and used RNA-seq to detect the expression levels of BrNRT2 genes after drought stress (Figure 7B).Three genes (BrNRT2.1,BrNRT2.3, and BrNRT2.10)with high expression levels in B. rapa were selected for further qRT-PCR detection (Figure 7C).The relative expression of BrNRT2.1 peaked after 4 h of drought treatment and then decreased.The relative expression of BrNRT2.3 was upregulated significantly at all time points, and its expression was 4-7-fold higher under drought stress relative to the CK treatment.The relative expression of BrNRT2.10 initially increased and then decreased, reaching a maximum after 4 h of drought treatment.

Effect of Salt Stress on the Expression of BrNRT2s
Salt stress can also negatively affect the yield and quality of B. rapa.Based on the results of the RNA-seq assay of the NRT2 genes in B. rapa following exposure to salt stress (Figure 8A), we selected three genes (BrNRT2.1,BrNRT2.3, and BrNRT2.8)with high expression levels in B. rapa for qRT-PCR detection (Figure 8B).The relative expression levels of BrNRT2.1 and BrNRT2.8 were significantly upregulated at each time point after treatment, and this was consistent with the results of the RNA-seq analysis.The relative expression of BrNRT2.3 first increased and then decreased, reaching a peak after 4 h of salt treatment.Overall, the pronounced changes in the expression of NRT2 members under salt stress suggest that there was a relationship between plant responses to salt stress and nitrogen transportation.

Effect of Salt Stress on the Expression of BrNRT2s
Salt stress can also negatively affect the yield and quality of B. rapa.Based on the results of the RNA-seq assay of the NRT2 genes in B. rapa following exposure to salt stress (Figure 8A), we selected three genes (BrNRT2.1,BrNRT2.3, and BrNRT2.8)with high expression levels in B. rapa for qRT-PCR detection (Figure 8B).The relative expression levels of BrNRT2.1 and BrNRT2.8 were significantly upregulated at each time point after treatment, and this was consistent with the results of the RNA-seq analysis.The relative expression of BrNRT2.3 first increased and then decreased, reaching a peak after 4 h of salt treatment.Overall, the pronounced changes in the expression of NRT2 members under salt stress suggest that there was a relationship between plant responses to salt stress and nitrogen transportation.

Protein Secondary Structure and Tertiary Structure Prediction of BrNRT2s
The structure of proteins is inextricably linked to their biological functions; thus, study of the structure of NRT2 proteins can provide important insights into their functions.In this study, we analyzed the predicted protein secondary structures of BrNRT2s and found that all members had α-helixes, random coil components, extended strands, and β-turns.The α-helixes accounted for the highest proportion of the protein secondary structures of BrNRT2s, and the β-turns accounted for the lowest proportion of protein secondary structures (Figure 9A).The different proportions of protein secondary structures in different family members might be related to the diverse roles of BrNRT2 genes in cell metabolism.The SWISS-MODEL analysis revealed that members of the same subgroup exhibit high structural similarity in protein tertiary structure (Figure 9B), indicating that they maintained homologous structures during the evolutionary process; this provides basic information that could aid subsequent studies of the functions of BrNRT2 proteins.

Protein Secondary Structure and Tertiary Structure Prediction of BrNRT2s
The structure of proteins is inextricably linked to their biological functions; thus, study of the structure of NRT2 proteins can provide important insights into their functions.In this study, we analyzed the predicted protein secondary structures of BrNRT2s and found that all members had α-helixes, random coil components, extended strands, and β-turns.The α-helixes accounted for the highest proportion of the protein secondary structures of BrNRT2s, and the β-turns accounted for the lowest proportion of protein secondary structures (Figure 9A).The different proportions of protein secondary structures in different family members might be related to the diverse roles of BrNRT2 genes in cell metabolism.The SWISS-MODEL analysis revealed that members of the same subgroup exhibit high structural similarity in protein tertiary structure (Figure 9B), indicating that they maintained homologous structures during the evolutionary process; this provides basic information that could aid subsequent studies of the functions of BrNRT2 proteins.

Prediction of BrNRT2 Protein Transmembrane Domains
We used TMHMM to predict the transmembrane domains of NRT2 proteins in B. rapa (Figure 10).Only the BrNRT2.9protein contained 13 transmembrane domains, and the remaining 13 BrNRT2 proteins had 8-11 transmembrane domains, suggesting that BrNRT2.9 can respond more quickly to environmental changes and has a stronger nitrate transport function.Additionally, the number of aa and their positions in each transmembrane domain are similar, indicating that they have similar structures and functions.

Prediction of BrNRT2 Protein Transmembrane Domains
We used TMHMM to predict the transmembrane domains of NRT2 proteins in B. rapa (Figure 10).Only the BrNRT2.9protein contained 13 transmembrane domains, and the remaining 13 BrNRT2 proteins had 8-11 transmembrane domains, suggesting that BrNRT2.9 can respond more quickly to environmental changes and has a stronger nitrate transport function.Additionally, the number of aa and their positions in each transmembrane domain are similar, indicating that they have similar structures and functions.

Analysis of the Protein Interaction Network of BrNRT2s
Proteins seldom perform their functions alone but interact with other proteins present in their surroundings to accomplish their biological activities [44].Therefore, understanding protein interactions is necessary for elucidating the mechanisms underlying cellular functions.B. rapa and A. thaliana are closely related, and the functions of the AtNRT2 genes have been intensively studied.Therefore, we could predict the function of corresponding homologous genes in B. rapa through a PPI analysis of the AtNRT2 genes, which would help further clarify the functions of BrNRT2 family members.We found that the relative expression of BrNRT2.3 was significantly upregulated after drought stress, and the relative expression of BrNRT2.1 and BrNRT2.8 was significantly upregulated after salt stress according to analysis of the qRT-PCR data.To clarify the interaction network of BrNRT2 proteins, integrated network maps of the homologous genes of BrNRT2.1,BrNRT2.3, and BrNRT2.8 and their interacting proteins were constructed based on the resources and algorithms available in the STRING database to identify their functions as well as physical interactions.The homolog of BrNRT2.3 in A. thaliana was AtNRT2.7, and the predicted protein interactions (Figure 11A) revealed strong interactions of AtNRT2.7 with nitrate reductase (NIA), nitrite reductase 1 (NIR1), and nitrate transporter 1 (NRT1).The homolog of BrNRT2.1 and BrNRT2.8 in A. thaliana was AtNRT2.6.AtNRT2.6 expresses high-affinity nitrate transporter proteins and sequentially interacts with nitrate reductase 1 (NIA1) to participate in nitrate assimilation; it also strongly interacted with nitrite reductase 1 (NIR1), nitrate transporter 1 (NRT1), and ammonium transporter (AMT) (Figure 11B).Some members of the NRT1 family have been shown to enhance drought and salt stress tolerance in plants.These findings suggest that BrNRT2s may interact with proteins in different families to respond to abiotic stress.

Analysis of the Protein Interaction Network of BrNRT2s
Proteins seldom perform their functions alone but interact with other proteins present in their surroundings to accomplish their biological activities [44].Therefore, understanding protein interactions is necessary for elucidating the mechanisms underlying cellular functions.B. rapa and A. thaliana are closely related, and the functions of the AtNRT2 genes have been intensively studied.Therefore, we could predict the function of corresponding homologous genes in B. rapa through a PPI analysis of the AtNRT2 genes, which would help further clarify the functions of BrNRT2 family members.We found that the relative expression of BrNRT2.3 was significantly upregulated after drought stress, and the relative expression of BrNRT2.1 and BrNRT2.8 was significantly upregulated after salt stress according to analysis of the qRT-PCR data.To clarify the interaction network of BrNRT2 proteins, integrated network maps of the homologous genes of BrNRT2.1,BrNRT2.3, and BrNRT2.8 and their interacting proteins were constructed based on the resources and algorithms available in the STRING database to identify their functions as well as physical interactions.The homolog of BrNRT2.3 in A. thaliana was AtNRT2.7, and the predicted protein interactions (Figure 11A) revealed strong interactions of AtNRT2.7 with nitrate reductase (NIA), nitrite reductase 1 (NIR1), and nitrate transporter 1 (NRT1).The homolog of BrNRT2.1 and BrNRT2.8 in A. thaliana was AtNRT2.6.AtNRT2.6 expresses high-affinity nitrate transporter proteins and sequentially interacts with nitrate reductase 1 (NIA1) to participate in nitrate assimilation; it also strongly interacted with nitrite reductase 1 (NIR1), nitrate transporter 1 (NRT1), and ammonium transporter (AMT) (Figure 11B).Some members of the NRT1 family have been shown to enhance drought and salt stress tolerance in plants.These findings suggest that BrNRT2s may interact with proteins in different families to respond to abiotic stress.

Discussion
Nitrate is an essential element for plant growth, as it serves as a key nutrient in the nitrogen assimilation pathway and as a vital signal for plant development [45].NRT2 family members are categorized as the high-affinity transport system (HATS) and are known to play key roles in nitrate uptake, transport, and response to biotic and abiotic stresses in various plant species.However, the NRT2 gene family have not been well studied in B. rapa.In this study, we analyzed 14 NRT2 genes from B. rapa, including their physicochemical properties, structural features, phylogenetic relationships, cis-elements, GO enrichment, expression patterns under abiotic stress, protein structures, and protein interactions.These results aided the analysis of their gene functions.
Phylogenetic relationships and collinearity analysis indicated close evolutionary relationships between BrNRT2s and AtNRT2s.Therefore, we could predict the gene functions of BrNRT2s based on the gene functions of AtNRT2s.BrNRT2.1,BrNRT2.3, and BrNRT2.8only contained one intron; other BrNRT2 genes had more introns.Previous studies have suggested that the expression levels of genes with fewer introns can rapidly change in response to stress [46].Moreover, conserved domain and motif analyses were carried out to clarify the relationship between the various members and their potential functions.All NRT2 family members have PLN00028 domain, indicating that the NRT2 subfamily might function through the PLN00028 domain.Motif 1, Motif 2, Motif 4, and Motif 5 are present in all BrNRT2 members, suggesting that they play an essential role in mediating nitrate transport.
Promoters are the regulatory centers of gene transcription, and an in-depth study of promoters can help clarify the mechanisms underlying the regulation of gene transcription.Analysis of the cis-elements in the promoter regions of BrNRT2 genes revealed abundant light-responsive elements, suggesting that the expression of BrNRT2 genes might be closely related to the regulation of light.In addition, many phytohormone-responsive elements and stress-responsive elements have been identified, suggesting that BrNRT2 genes may be involved in growth and developmental activities and stress responses in B. rapa.GO analysis of the BrNRT2 genes revealed that the enriched terms in BP were mainly related to nitrate transport, cellular response to nitrate, and nitrate assimilation.Analysis

Discussion
Nitrate is an essential element for plant growth, as it serves as a key nutrient in the nitrogen assimilation pathway and as a vital signal for plant development [45].NRT2 family members are categorized as the high-affinity transport system (HATS) and are known to play key roles in nitrate uptake, transport, and response to biotic and abiotic stresses in various plant species.However, the NRT2 gene family have not been well studied in B. rapa.In this study, we analyzed 14 NRT2 genes from B. rapa, including their physicochemical properties, structural features, phylogenetic relationships, cis-elements, GO enrichment, expression patterns under abiotic stress, protein structures, and protein interactions.These results aided the analysis of their gene functions.
Phylogenetic relationships and collinearity analysis indicated close evolutionary relationships between BrNRT2s and AtNRT2s.Therefore, we could predict the gene functions of BrNRT2s based on the gene functions of AtNRT2s.BrNRT2.1,BrNRT2.3, and BrNRT2.8only contained one intron; other BrNRT2 genes had more introns.Previous studies have suggested that the expression levels of genes with fewer introns can rapidly change in response to stress [46].Moreover, conserved domain and motif analyses were carried out to clarify the relationship between the various members and their potential functions.All NRT2 family members have PLN00028 domain, indicating that the NRT2 subfamily might function through the PLN00028 domain.Motif 1, Motif 2, Motif 4, and Motif 5 are present in all BrNRT2 members, suggesting that they play an essential role in mediating nitrate transport.
Promoters are the regulatory centers of gene transcription, and an in-depth study of promoters can help clarify the mechanisms underlying the regulation of gene transcription.Analysis of the cis-elements in the promoter regions of BrNRT2 genes revealed abundant light-responsive elements, suggesting that the expression of BrNRT2 genes might be closely related to the regulation of light.In addition, many phytohormone-responsive elements and stress-responsive elements have been identified, suggesting that BrNRT2 genes may be involved in growth and developmental activities and stress responses in B. rapa.GO analysis of the BrNRT2 genes revealed that the enriched terms in BP were mainly related to nitrate transport, cellular response to nitrate, and nitrate assimilation.Analysis of tissue-specific data showed that BrNRT2 genes were mainly expressed in the roots, which highlights their importance in nitrate uptake and transport.
An increasing number of studies have shown that nitrogen uptake, transport, and assimilation are related to drought and salt tolerance in plants [27,29].Therefore, the expression of BrNRT2 genes has been observed under drought and salt stress conditions.The qRT-PCR results showed that the relative expression of BrNRT2.3 was significantly upregulated after drought stress.Gene structure analysis showed that BrNRT2.3contains only one intron, and the expression level of this gene can change rapidly in response to stress.Cis-element analysis showed that BrNRT2.3contains ABA-responsive elements and MeJA-responsive elements.ABA is a critical hormone that regulates water status and stomatal movement.Under drought conditions, ABA production and accumulation in plant guard cells induce the closure of the stomata to conserve water [47].MeJA can induce the synthesis of defensive compounds that improve drought resistance by altering various biochemical characteristics of plants, such as increasing the concentration of organic osmoprotectants and oxidase activities [48,49].BrNRT2.3 is mainly expressed in the stem and leaf tissues, suggesting that these genes might play a role in the long-distance transport of nitrate from the roots to the aboveground parts.Protein-protein interaction predictions also suggest that the BrNRT2.3homolog AtNRT2.7 might interact with AtNRT1.1,AtNRT1.5, AtNRT1.6,AtNRT1.7, and AT2G26690.AtNRT1.1 (CHL1) can regulate stomatal opening; a previous study showed that chl1 mutant plants are drought tolerant because of their ability to reduce water loss [50].AtNRT1.5 mediates the redistribution of nitrate to the root system and promotes the expression of stress-response-related genes, which enhances salt, drought, and cadmium stress tolerance.The redistribution of nitrate within plants serves as a signal that links diverse stress cues to extensive physiological adjustments, which ultimately enhances their stress tolerance [51].AtNRT1.6,AtNRT1.7, and AT2G26690 all belong to the PTR2/POT transporter family, and PTR2 in A. thaliana is negatively regulated by ABI4 and promotes water uptake during early seed germination [52].Similarly, BrNRT2.3 may help respond to drought stress by interacting with genes in these families.Therefore, we speculate that when plants initially experience water deficits, their main response is to limit water loss and enhance water uptake; however, when drought persists, BrNRT2.3might mediate the rapid response to changes in the external environment and cope with drought stress through the hormone signaling pathway or by interacting with other proteins.
In addition, we found that BrNRT2.1 and BrNRT2.8 may play a role in regulating salt stress.Both BrNRT2.1 and BrNRT2.8contain only one intron, and the expression levels of these genes can change rapidly in response to stress.Cis-element analysis revealed that BrNRT2.1 contains ABA-responsive elements and auxin-responsive elements, and BrNRT2.8contains MeJA-responsive elements, gibberellin-responsive elements, and ABAresponsive elements; all four hormones play a regulatory role in the response to salt stress.Auxin affects gene expression through a series of functionally distinct transcription factors, including DNA-binding auxin response factors (ARFs).Different ARFs regulate the soluble sugar content and maintain the chlorophyll content to promote the adaptation of plants to salt stress [53].In rapeseed (Brassica napus L. cv.Talaye), the exogenous application of MeJA increased the soluble sugar level, relative water content, and photosynthetic rate to counteract the inhibitory effect of NaCl [54].In sorghum, the exogenous application of gibberellins (GAs) can alleviate salt-stress-induced cell wall thickening by increasing the cellulose and hemicellulose content of root cells, allowing the rapid entry of water into root cells, and altering the dynamic balance of endogenous hormones in cells, thereby mitigating the effects of salt stress on germination and seedling growth [55].Under highsalt conditions, ABA can stimulate short-term responses such as stomatal closure, thereby maintaining the water balance and mediating long-term growth responses by regulating the expression of stress-response genes [56].Tissue-specific analysis showed that BrNRT2.1 and BrNRT2.8 are highly expressed in the roots, indicating that these genes may play an important role in the uptake of nitrate from the soil and in coping with root-associated stresses.Both the transcriptome data and qRT-PCR results showed that the relative expression of BrNRT2.1 and BrNRT2.8 was significantly elevated under salt stress.The predicted protein interactions also indicated that the BrNRT2.1 and BrNRT2.8homolog AtNRT2.6

Figure 1 .
Figure 1.Chromosomal localization of BrNRT2s.The chromosomal position of each NRT2 gene was mapped according to the B. rapa genome.The number of chromosomes is listed on the left side of each chromosome.Each chromosome shows gene density.

Figure 1 .
Figure 1.Chromosomal localization of BrNRT2s.The chromosomal position of each NRT2 gene was mapped according to the B. rapa genome.The number of chromosomes is listed on the left side of each chromosome.Each chromosome shows gene density.

Figure 3 .
Figure 3. Gene structure and conserved domain analysis of BrNRT2s.(A) Analysis of gene structure.The introns and exons are shown as black lines and blue boxes, respectively.(B) Analysis of conserved domains of BrNRT2 genes.(C) Analysis of the conserved motifs of BrNRT2 genes.The length of each motif is also shown.(D) Motif sequence.

Figure 3 .
Figure 3. Gene structure and conserved domain analysis of BrNRT2s.(A) Analysis of gene structure.The introns and exons are shown as black lines and blue boxes, respectively.(B) Analysis of conserved domains of BrNRT2 genes.(C) Analysis of the conserved motifs of BrNRT2 genes.The length of each motif is also shown.(D) Motif sequence.

Figure 4 .
Figure 4. Predicted cis-elements in BrNRT2 promoters.The figure shows the number of cis-elements contained in the BrNRT2 promoters.

Figure 4 .
Figure 4. Predicted cis-elements in BrNRT2 promoters.The figure shows the number of cis-elements contained in the BrNRT2 promoters.

Genes 2023 , 19 Figure 5 .
Figure 5. GO enrichment analysis of BrNRT2s.The size and color of the dot bubbles indicate the number of genes and p-value, respectively.

Figure 5 .Figure 6 .
Figure 5. GO enrichment analysis of BrNRT2s.The size and color of the dot bubbles indicate the number of genes and p-value, respectively.

Figure 6 .
Figure 6.Expression patterns of BrNRT2s in different tissues.The abundance of each gene was determined using TPM.

Genes 2023 , 19 Figure 7 .
Figure 7. Expression analysis of BrNRT2s under drought stress.(A) Analysis of transcriptome data of NRT2 genes under drought stress in drought-sensitive B. rapa obtained from the Brassicaceae Database.(B) Heatmap of expression patterns of BrNRT2 genes under drought stress.(C) The relative expression levels of BrNRT2 genes under drought stress were analyzed using qRT-PCR.

Figure 7 .
Figure 7. Expression analysis of BrNRT2s under drought stress.(A) Analysis of transcriptome data of NRT2 genes under drought stress in drought-sensitive B. rapa obtained from the Brassicaceae Database.(B) Heatmap of expression patterns of BrNRT2 genes under drought stress.(C) The relative expression levels of BrNRT2 genes under drought stress were analyzed using qRT-PCR.

Figure 8 .
Figure 8. Expression analysis of BrNRT2s under salt stress.(A) Heatmap of expression patterns of BrNRT2 genes under salt stress.(B) The relative expression levels of BrNRT2 genes under salt stress were analyzed using qRT-PCR.

Figure 8 .
Figure 8. Expression analysis of BrNRT2s under salt stress.(A) Heatmap of expression patterns of BrNRT2 genes under salt stress.(B) The relative expression levels of BrNRT2 genes under salt stress were analyzed using qRT-PCR.

Figure 9 .
Figure 9.Protein secondary and tertiary structure prediction of BrNRT2s.(A) Prediction of the secondary structure of BrNRT2 proteins.Different colors indicate different secondary structures.(B) Prediction of the tertiary structures of BrNRT2 proteins.Different colors indicate different subunits.

Figure 9 .
Figure 9.Protein secondary and tertiary structure prediction of BrNRT2s.(A) Prediction of the secondary structure of BrNRT2 proteins.Different colors indicate different secondary structures.(B) Prediction of the tertiary structures of BrNRT2 proteins.Different colors indicate different subunits.

Figure 10 .
Figure 10.Prediction of BrNRT2 protein transmembrane domains.The gene names are listed above, the x-axis shows the length of the protein sequence, and the y-axis shows the probability.

Figure 10 .
Figure 10.Prediction of BrNRT2 protein transmembrane domains.The gene names are listed above, the x-axis shows the length of the protein sequence, and the y-axis shows the probability.

Figure 11 .
Figure 11.Predicted interaction network of NRT2s.(A) AtNRT2.7 (BrNRT2.3homologous gene) PPIs.(B) AtNRT2.6 (BrNRT2.1 and BrNRT2.8homologous gene) PPIs.The colored nodes represent the query proteins.The filled nodes represent proteins with known or predicted 3D structures.The edges represent protein associations.Different colored lines between the nodes represent the type of evidence for the interaction.

Figure 11 .
Figure 11.Predicted interaction network of NRT2s.(A) AtNRT2.7 (BrNRT2.3homologous gene) PPIs.(B) AtNRT2.6 (BrNRT2.1 and BrNRT2.8homologous gene) PPIs.The colored nodes represent the query proteins.The filled nodes represent proteins with known or predicted 3D structures.The edges represent protein associations.Different colored lines between the nodes represent the type of evidence for the interaction.

Table 1 .
Information of NRT2 family genes in B. rapa.

Table 1 .
Information of NRT2 family genes in B. rapa.